Phospho s6

Cells were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP-40; 50 mM NaF with protease inhibitors) and incubated on ice for 30 min. Lysates were centrifuged at 20,817g at 4 °C for 10 min and supernatant was collected. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein samples were mixed with SDS Laemmli loading buffer, boiled and electrophoresed using NuPAGE Bis-Tris Gels (Life Technologies), then transferred onto PVDF membranes (Millipore). Blocking was performed for 45 min using TBST supplemented with 5% non-fat dry milk and blotting performed with primary antibodies at 4 °C for 16 h. The following antibodies were used: ADSL (Abcam #ab154182), GMPS (Cell Signaling #14602), PRPS1 (Abcam #ab154721), MYC (N-262, Santa Cruz #sc-764), total AKT (Cell Signaling #4691), phospho-AKT (S473, Cell Signaling #9271), total S6 (Cell Signaling #2317), phospho-S6 (235/236, Cell Signaling #4858), phospho-S6 (240/242, Cell Signaling #5364), total p70 S6K (Cell Signaling #2708), phospho-p70 S6K (Cell Signaling #9234) and α-tubulin (Sigma #T6074). All antibody validation information is available in the product’s manual. For western blotting, the dilution was 1:500 for MYC antibody and 1:1,000 for all other antibodies.

Tissues were fixed in 10% formalin overnight and embedded in paraffin. Immunohistochemical (IHC) and immunofluorescence (IF) staining was performed as previously described^{11 (link),14 (link)}. IHC slides were scanned with Pannoramic Digital Slide Scanner (3DHISTECH) and images were cropped from virtual slides in Pannoramic Viewer. IF slides were imaged with Nikon A1R Confocal Laser Microscope and quantified with ImageJ. Primary antibodies used include CK5 (Covance, PRB-160P), CK8 (Covance, MMS-162P), Ki67 (Fisher, RM-9106-S1), cleaved caspase 3 (Cell Signaling Technology, 9661), Gr-1 (BioLegend, 108401), phospho-S6 (Cell Signaling Technology, 4858). For Western blot analysis, cells or fresh tissues were lysed on ice using RIPA buffer (Boston BioProducts) supplemented with protease and phosphatase inhibitors (Roche). Western blot procedure was performed as previously described^{11 (link),14 (link)}. Primary antibodies used include phospho-Met (Cell Signaling Technology, 3077), phospho-VEGFR2 (Cell Signaling Technology, 3770), phospho-Erk1/2 (Cell Signaling Technology, 4370), phospho-Akt (Cell Signaling Technology, 4060), phospho-mTOR (Cell Signaling Technology, 5536), phospho-p70 S6K (Cell Signaling Technology, 9234), phospho-S6 (Cell Signaling Technology, 4856), and vinculin (Millipore, 05-386).